1. Field of the Invention
The present invention relates to parathyroid hormone-related peptide (PTHrP) analogs. In particular, the invention relates to PTHrP analogs having one or more amino acid substitutions that confer PTH-2 receptor antagonist or agonist properties to the analog.
2. Description of Related Art
Parathyroid hormone (PTH) is a major regulator of calcium homeostasis whose principal target cells occur in bone and kidney. Regulation of calcium concentration is necessary for the normal function of the gastrointestinal, skeletal, neurologic, neuromuscular, and cardiovascular systems. PTH synthesis and release are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood at three sites of calcium exchange: gut, bone, and kidney. PTH contributes to net gastrointestinal absorption of calcium by favoring the renal synthesis of the active form of vitamin D. PTH promotes calcium resorption from bone by inhibiting osteoblasts and, indirectly, by stimulating differentiation of the bone-resorbing cells, osteoclasts. It also mediates at least three main effects on the kidney: stimulation of tubular calcium reabsorption, enhancement of phosphate clearance, and promotion of an increase in the enzyme that completes synthesis of the active form of vitamin D. PTH exerts these effects primarily through receptor-mediated activation of adenylate cyclase, although receptor-mediated activation of phospholipase C by PTH has also been reported (Hruska et al., J. Clin. Invest. 79:230 (1987)).
Disruption of calcium homeostasis may produce many clinical disorders (e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy, and neuropathy) and usually results from conditions that produce an alteration in the level of parathyroid hormone. Hypercalcemia is a condition that is characterized by an elevation in the serum calcium level. It is often associated with primary hyperparathyroidism in which an excess of PTH production occurs as a result of a lesion (e.g., adenoma, hyperplasia, or carcinoma) of the parathyroid glands. Another type of hypercalcemia, humoral hypercalcemia of malignancy (HHB) is the most common paraneoplastic syndrome. It appears to result in most instances from the production by tumors (e.g., squamous, renal, ovarian, or bladder carcinomas) of a novel class of protein hormone which shares amino acid homology with PTH. These PTH-related proteins (PTHrP) appear to mimic certain of the renal and skeletal actions of PTH and are believed to interact with the PTH receptor in these tissues. PTHrP is normally found at low levels in many tissues, including keratinocytes, brain, pituitary, parathyroid, adrenal cortex, medulla, fetal liver, osteoblast-like cells, and lactating mammary tissues. In many HHM malignancies, PTHrP is found in the circulatory system at high levels, thereby producing the elevated calcium levels associated with HHM.
The pharmacological profiles of PTH and PTHrP are nearly identical in most in vitro assay systems, and elevated blood levels of PTH (i.e., primary hyperparathyroidism) or PTHrP (i.e., HHM) have comparable effects on mineral ion homeostasis (Broadus, A. E. & Stewart, A. F., "Parathyroid hormone-related protein: Structure, processing and physiological actions," in Basic and Clinical Concepts, Bilzikian, J. P. et al., eds., Raven Press, New York (1994), pp. 259-294; Kronenberg, H. M. et al., "Parathyroid hormone: Biosynthesis, secretion, chemistry and action," in Handbook of Experimental Pharmacology, Mundy, G. R. & Martin, T. J., eds., Springer-Verlag, Heidelberg (1993), pp. 185-201). The similarities in the biological activities of the two ligands can be explained by their interaction with a common receptor, the PTH/PTHrP receptor, which is expressed abundantly in bone and kidney (Urena, P. et al., Endocrinology 134:451-456 (1994)).
The binding of either radiolabeled PTH-(1-34) or PTHrP-(1-36) to the PTH/PTHrP receptor is competitively inhibited by either unlabeled ligand (Juppner, H. et al., J. Biol. Chem. 263:8557-8560 (1988); Nissenson, R. A. et al., J. Biol. Chem. 263:12866-12871 (1988)). Thus, the recognition sites for the two ligands in the PTH/PTHrP receptor probably overlap. In both PTH and PTHrP, the 15-34 region contains the principal determinants for binding to the PTH/PTHrP receptor. Although these regions show only minimal sequence homology (only 3 amino acid identities), each 15-34 peptide can block the binding of either PTH-(1-34) or PTHrP-(1-34) to the PTH/PTHrP receptor (Nussbaum, S. R. et al., J. Biol. Chem. 255:10183-10187 (1980); Caulfield, M. P. et al., Endocrinology 127:83-87 (1990); Abou-Samra, A.-B. et al., Endocrinology 125:2215-2217 (1989)). Further, the amino terminal portion of each ligand is required for bioactivity, and these probably interact with the PTH/PTHrP receptor in similar ways, since 8 of 13 of these residues are identical in PTH and PTHrP.
The PTH/PTHrP receptor is a member of a distinct family of G protein-coupled receptors (Juppner, H. et al., Science 254:1024-1026 (1991); Abou-Samra, A. B. et al., Proc. Natl. Acad. Sci (USA) 89:2732-2736 (1992)) that includes receptors for other peptide hormones such as secretin (Ishihara, T. et al., EMBO J. 10:1635-1641 (1991)), calcitonin (Lin, H. Y. et al., Science 254:1022-1024 (1991)) and glucagon (Jelinek, L. J. et al., Science 259:1614-1616 (1993)). Using degenerate oligonucleotides corresponding to conserved regions of the PTH/secretin/calcitonin receptor family, Usdin et al. has identified a new receptor cDNA derived from rat and human brain that was most closely related to the PTH/PTHrP receptor (51% overall amino acid sequence identity) (Usdin, T. et al., J. Biol. Chem. 270:15455-15458 (1995)). This new receptor, the PTH-2 receptor, responded efficiently, potently, and specifically to PTH-(1-34), but interestingly, did not respond at all to PTHrP. Id. This observation implied that structural differences in the PTH and PTHrP ligands determined selectivity for interaction with the PTH-2 receptor. The PTH-2 receptor was found predominantly in the brain and pancreas. Id.
Since the PTH-2 receptor was found in brain and pancreas, it is likely to have a physiological role in the normal functioning of those organs. Diseases associated with brain and pancreatic dysfunction may in fact be explained by the altered action of the PTH-2 receptor. Such diseases could then be treated with a PTH-2 receptor selective antagonist. Other antagonists that are available are not selective and thus, are not desired because they would also antagonize the PTH/PTHrP receptor, which could have negative consequences in terms of calcium regulation and skeletal function.
Accordingly, there is a need in the art for the development of PTH-2 receptor selective agonists and antagonists: (1) to assist in further elucidating the biological role of the PTH-2 receptor; (2) to map specific sites of ligand-receptor interaction; and (3) as potential new therapeutic compositions that can be used in the treatment of disorders associated with altered action or genetic mutation of the PTH-2 receptor.